The long-range goals of this project are to dissect the regulatory events controlling heme biosynthesis during the maturation of erythroid progenitor cells with emphasis on the gene encoding the second enzyme of the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALA-D). Four specific aims are set. First, erythroid colony-forming units (CFU-E) will be purified from thiamphenicol-treated, anemic mice and subsequently cultured to obtain samples from various stages of erythropoiesis. Enzyme assays for the first four and the last enzyme of the heme biosynthetic pathway will be performed to determine the rate-limiting steps in erythroid cells. Concurrent quantitation of changing mRNA levels by RNase protection assays will identify which of the rate-limiting steps are controlled at the transcriptional level. Second, the erythroid-specific transcriptional promoter of the ALA-D gene will be analyzed in detail using gel-retardation assays and DNaseI footprinting to locate nucleotide sequences which bind proteins in vitro. Third, the sequences identified in the previous section will be functionally tested by transient transfection assays in murine erythroleukemia cells and crucial sequences identified by loss of function following in vitro mutagenesis. Fourth, characterization of the ALA-D gene dosage polymorphism found in the inbred mouse will be extended to wild caught mice. The molecular structure of the duplicated and triplicated loci will be determined and analyzed at the nucleotide sequence level and a model will be built for the mechanism of increased ALA-D expression by recombination and gene duplication which may be applicable to other regions of the genome.